Selenophosphate synthetase forms the energy-rich compound, seleno-phosphate, from ATP and selenide. In place of an essential cysteine residue in the E. coli enzyme, enzymes from mouse embryo and Methanococcus jannashii (an extreme thermophile) contain selenocysteine, in human and rat there is a threonine residue, and in Drosophil, there is arginine. To evaluate the relative catalytic activities of enzymes having Se, S, OH, or NH in the active site, purified enzymes are required. Wild type and a Cys mutant form of the mouse embryo enzyme expressed in insect cells, the Secys enzyme produced in M. jannashii grown at 65-80degreesC, and the human enzyme expressed in E. coli are available to our group. If, as suspected, free selenide is inactive in vitro as substrate for the threonine enzyme, then this enzyme can be used as an assay for detection and identification of the natural in vivo selenium substrate, such as a perselenide type of compound. In continuing studies on selenoproteins produced by Methanococcus vannielii, additional amounts of a 33 kDa protein subunit have been prepared for tryptic peptide sequence determination. The sequence of the first 63 amino acid residues from the N-terminus of this protein already determined was not sufficient to establish identity. Correlation of cellular levels of this protein with obligatory use of urate as nitrogen source for growth of M. vannielii suggests its identity as a subunit of xanthine dehydrogenase. Other 75Se-labeled and unlabeled protein bands isolated and subjected to partial N-terminal sequencing from these preparations were identified as follows: The N-terminal sequence of a 50 kDa 75Se-labeled protein band corresponded to the alpha subunit of the F420 hydrogenase of M. jannashii and also a subunit of Methanococcus voltae F420 hydrogenase. This should be the selenocysteine- containing subunit of the M. vannielii enzyme purified by S. Yamazaki in this laboratory. The N-terminal sequence of a 30 kDa protein matched the sequence of the gamma subunit of M. jannashii F420 hydrogenase and also a subunit of the enzyme from M. voltae. The N-terminal sequence of a 42 kDa protein band on the SDS gels was homologous to the delta subunit of M. jannashii F420 hydrogenase and also to a subunit of M. voltae. These partial sequences of subunits of M. vannielii F420 reducing hydrogenase are to be deposited in the appropriate gene banks for use of other investigators.